RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.

M. PHARM SYNOPSIS

YEAR OF ADMISSION-JULY 2011

TITLE OF THE SYNOPSIS

“AN INVESTIGATION OF ANTI-EPILEPTIC AND ANTI-PSYCHOTIC ACTIVITIES OF SHILAJIT IN EXPERIMENTAL ANIMALS"

BYMr. SHARANBASAPPA

M. PHARM., PART-IDEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Mr. SYED MANSOOR AHMED M.Pharm., (Ph.D) Asst. Professor

DEPARTMENT OF PHARMACOLOGY

INSTITUTIONSREE SIDDAGANGA COLLEGE OF PHARMACY

B. H. ROAD, TUMKUR-572 102. KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE AND ADDRESS

Mr. SHARANBASAPPA

IST M.Pharma.Department of Pharmacology,

Sree Siddaaganga College of Pharmacy,B.H.Road, Tumkur – 572 102

2. NAME OF THE GUIDE Mr. SYED MANSOOR AHAMEDM. Pharm.,(Ph.D)

Assistant Professor ,Department of Pharmacology.

3. NAME OF THE INSTITUTION

SREE SIDDAGANGA COLLEGE OF PHARMACYB. H. ROAD, TUMKUR- 572 102

4. COURSE OF STUDY AND SUBJECT

MASTER OF PHARMACY (PHARMACOLOGY)

5. DATE OF ADMISSION OF COURSE

23rd JULY 2011

6. TITLE OF THE TOPIC

“An investigation of anti-epileptic and anti-psychotic activities of

Shilajit in experimental animals”

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7.0 BRIEF RESUME OF THE INTENDED WORK

7.1 - NEED FOR THE STUDY

The term Epilepsy, based on the Greek word “epilambaein” meaning “to seize” has

been first mentioned by Hippocrates.1 Recognised from the drawn of history as

‘disease of lightening’, it was correctly described by JH Jackson over a century ago.2

Epilepsy is one of the most common chronic neurological disorders with no age, racial,

social, sexual or geographical boundaries and affects about 50 million people

worldwide.3 The prevalence is high in tropical countries, particularly in Africa, where

it varies between 10 and 55 per 1000, with an estimated mean prevalence of 15.4 People

with epilepsy are at special risk for two life-threatening conditions: status epilepticus

and sudden unexplained death. 5

The term Psychosis, based on the Greek word “psyche” for mind/soul, and “-osis”, for

abnormal condition; means abnormal condition of the mind, and is a generic

psychiatric term for a mental state often described as involving a “loss of contact with

reality”. People suffering from psychosis are described as psychotic. Psychosis is given

to the more severe forms of psychiatric disorder, during which hallucinations,

delusions and impaired insight may occur. 6

Epilepsy and psychosis are among the disorders that are strongly associated with

significant psychological and social consequences for everyday living. CNS (Central

Nervous System) disorders often play a major role and will be a growing cause of

primary and secondary disability in the next 10 years. 7 According to WHO report one

in four people in the world will be affected by mental or neurological disorders at some

point in their lives. Around 450 million people currently suffer from such conditions,

placing mental disorders among the leading causes of ill-health and disability

worldwide. 8

Most CNS ailments follow a complex biology, and can have differing outcomes

depending on predisposing factors. 10 Though the available drugs fulfill the

requirements in the segment, there is a need of more effective drugs that are better

tolerated and cost effective to enhance long-term compliance. These diseases involve a

change in the basic physiology of the nervous system. It is difficult or sometimes

3

impossible to reverse these changes. Hence, the drugs are more palliative as compared

to curative. Besides, several neurological ailments are not entirely understood, hence

the treatments are not adequate, and even if they are well understood, therapy is

associated with limitations. 9

Many of the drugs merely treat the symptoms and do not provide cures. Current anti-

epileptic drugs are effective in controlling seizures in about 70% of patients, but their

use is often limited by side effects. 10 Hence there is a need for development of cheap,

effective and safe agents from plant, animals and mineral sources. The development of

new therapies has the potential to provide patients with significant improvements. In

folk medicine, Shilajit has been used to treat diverse clinical conditions ranging from

anaemia to nervous disorders. 11 Hence, the present project has been undertaken for

screening of the herbo-mineral drug Shilajit, 12 for anti-epileptic and anti-psychotic

activity in experimental animals, as Shilajit has not been studied for its anti-epileptic

and anti-psychotic activity despite of its use in treatment of nervous disorders in

traditional medicines. 11

7.2 REVIEW OF LITERATURE

Shilajit is a herbo-mineral drug ejected out of fissures in iron rich rocks, during hot

weather. 12

It is found to be produced naturally in mountainous areas, especially Himalayas,

Vindhya and other mountains in India and also in Nepal. It may also be found as a tar

in the earth crust, formed due to decomposition of vegetable substances. It is observed

that ‘Momia’ which occurs in Arabia and Persian mountains, resembles Shilajit. 12

The following forms of this drug have been reported in ancient Hindu literature, viz.

1. Iron Shilajit (blackish-brown variety)

2. Copper Shilajit (blue variety)

3. Silver Shilajit (white variety)

4. Gold Shilajit (red variety)

Synonyms

4

Sanskrit : Shilajit, Silajit, Silaras

Hindi : Silajita, Ral-yahudi

English : Asphalt, Mineral Pitch, Jew’s Pitch, Vegetable Asphalt

Latin : Asphaltum

Tamil : Perangyum, Uerangyum

Bengali : Silajatu

Gujarati : Silajita

In Sanskrit, Shilajit means ‘winner of rock’ (Mukherjee 1992). Another meaning is

“sweat of the rock” (Tirtha 1998).

Shilajit is a blackish-brown exudation, of variable consistency, obtained from steep

rocks of different formations found in the Himalayas at altitudes “between” 1000 to

5000 meter, from Arunachal Pradesh in the east to Kashmir in the west. It is also found

in Afghanistan, Nepal, Bhutan, Pakistan, China, Tibet and U.S.S.R. (Tien-shan, Ural

and Caucasus) (Jaiswal 1992). The black form of Shilajit is the most commonly used

medicinal form (Halpern 2003).

Medicinal uses

It is used as a general tonic stimulant, aphrodisiac 12, diabetes mellitus 13, nervous

disorders 11, obesity. 14 Shilajit of authentic quality is an important ingredient of

ayurvedic preparations meant for strengthening immunising system of human body. It

is a unique blend of herbal and mineral ingredients formed by a unique natural process.

Reported activities

Shilajit has been reported to be anti-anxiety 15, antioxidant 16, immuno-modulator 17,

asthma 18, adaptogenic agent 19, anti-ulcerogenic and anti-inflammatory. 20

7.3 OBJECTIVES OF THE STUDY

1. Collection of Shilajit from market as Shilajit powder.

2. To investigate the anti-epileptic activity of Shilajit by using maximal electro shock

induced seizures, isoniazid induced seizures and pentylenetetrazol induced seizures in

experimental animals.

3. To investigate the anti-psychotic activity of Shilajit by using apomorphine induced

stereotype behavior, pilocarpine induced purposeless chewing and apomorphine

induced climbing behavior in experimental animals.

MATERIALS AND METHODS

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8.0 8.1 Materials – List of Chemicals and Instruments used

Shilajit, Caffeine, Tween 80

Ethyl acetate, Chloroform

Phenytoin, Diazepam, Haloperidol

Apomorphine, Pentylenetetrazol

Pilocarpine, Mecamylamine

Electro convulsometer

Melting point apparatus

8.2 Experimental animals

Adult albino wistar rats of either sex weighing approximately 180-200 g will be used.

They will be housed in standard laboratory conditions and fed with standard animal

feed and will be given water ad libitum.

8.3 Dose of Shilajit

Shilajit will be administered at dose of 25 and 50 mg/kg. 15

8.4 Grouping of animals

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group.

Group I – control

Group II – suitable dose of standard

Group III – 25 mg/kg of Shilajit

Group IV – 50 mg/kg of Shilajit

8.5 Experimental procedure

8.51 Anti-epileptic study using maximal electro shock induced seizures

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The test will be started 60 minutes after oral treatment with

suitable doses of Shilajit or the standard. An electro convulsometer with corneal or ear

electrodes will be used to deliver the stimuli. The incidence and duration of extensor

tonus will be noted. A complete abolition of hind limb tonic extension will be

considered as 100% protection. 21

8.52 Anti-epileptic study using isoniazid induced seizures

6

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The groups will be treated with suitable doses of Shilajit or the

standard. 60 minutes after the treatment with Shilajit, animals will be injected with

subcutaneous doses of isoniazid. During the next 120 minutes the occurrence of clonic

seizures and tonic seizures will be recorded [17]. The percentage of seizures or deaths

occurring in the control group is taken as 100%. The suppression of these effects in

the treated group is calculated as percentage of controls.22

8.53 Anti-epileptic study using pentylenetetrazol (PTZ) induced seizures

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The groups will be treated with suitable doses of Shilajit or the

standard. 60 minutes after the treatment with Shilajit, animals will be injected with

subcutaneous doses of PTZ. During the next 120 minutes the occurrence of clonic

seizures, tonic seizures and death is required. The percentage of seizures or deaths

occurring in the control group is taken as 100%. The suppression of these effects in

the treated group is calculated as percentage of controls. 23

8.54 Anti-psychotic study using apomorphine induced stereotypy

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The suitable dose of Shilajit or the standard will be

administered 60 minutes prior apomorphine dosage. Apomorphine HCl will be injected

subcutaneous. The animals will be placed in individual plastic cages. A 10 second

observation period is used to measure the presence of stereotypic activity such sniffing,

licking and chewing 10 minutes after apomorphine administration. An animal is

considered protected if this behavior is reduced or abolished. 24

8.55 Anti-psychotic study using pilocarpine induced purposeless chewing

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The groups will be treated with suitable doses of Shilajit or the

standard. 60 minutes after the treatment with Shilajit, animals will be injected with i.p.

doses of pilocarpine. Then the animals will be placed individually in a large glass

cylinder (height 30 cm, diameter 20 cm) at 21 ± 1oC and allowed to habituate for 15

minutes before injection of drugs. Numbers of chews are counted by direct observation

immediately after drug administration. The results will be presented as numbers of

chews in a 30 minute period.25

8.56 Anti-psychotic study using apomorphine induced climbing behavior

7

The albino wistar rat will be randomly distributed into four groups containing six

animals in each group. The groups will be treated with suitable doses of Shilajit or the

standard. 60 minutes after the treatment with Shilajit, animals will be injected with

haloperidol i.p. Then the animals are placed individually in wire- mesh stick cages; 30

minutes after the treatment is given. 10, 20 and 30 minutes after apomorphine

administration, they are observed for climbing behavior and scored as follows:

0 = four paws on the floor

1 = forefeet holding the vertical bars

2 = forefeet holding the bars 22

8.6 Experimental design

8.61 Anti-epileptic activity (single and multiple dose study)

a. Maximal electro shock (MES) induced seizures

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

MES

Hind limb tonic extension

II Phenytoin (90 mg/kg, i.p.)

+ MES

III Shilajit (25 mg/kg, p.o.) +

MES

IV Shilajit (50 mg/kg, p.o.) +

MES

Multiple dose study is conducted for duration of 15 days.

b. Isoniazid (INH) induced seizures

8

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

INH (300 mg/kg, s.c.)

Occurrence of tonic –

clonic seizure

II Diazepam (4 mg/kg, i.p.) +

INH (300 mg/kg, s.c.)

III Shilajit (25 mg/kg, p.o.) +

INH (300 mg/kg, s.c.)

IV Shilajit (50 mg/kg, p.o.) +

INH (300 mg/kg, s.c.)

Multiple dose study is conducted for duration of 15 days. INH is administered on 15 th

day.

c. Pentylenetetrazol (PTZ) induced convulsions

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

PTZ (60 mg/kg, s.c.)

Occurrence of tonic –

clonic seizure

II Diazepam (4 mg/kg, i.p.) +

PTZ (60 mg/kg, s.c.)

III Shilajit (25 mg/kg, p.o.) +

PTZ (60 mg/kg, s.c.)

IV Shilajit (50 mg/kg, p.o.) +

PTZ (60 mg/kg, s.c.)

Multiple dose study is conducted for duration of 15 days. PTZ is administered on 15 th

day.

8.62 Antipsychotic activity (single and multiple dose study)

a. Apomorphine induced stereotype behavior

9

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

Apomorphine (1.5 mg/kg,

s.c.)

10

Stereotyped behavior

II Standard drug (1 mg/kg,

i.p.) + Apomorphine (1.5

mg/kg, s.c.)

III Shilajit (25 mg/kg, p.o.) +

Apomorphine (1.5 mg/kg,

s.c.)

IV Shilajit (50 mg/kg, p.o.) +

Apomorphine (1.5 mg/kg,

s.c.)

Multiple dose study is conducted for duration of 15 days. Apomorphine is administered

on 15th day.

b. Pilocarpine induced purposeless chewing

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

Pilocarpine (1mg/kg, i.p.)

Numbers of chewing in 30

minutes period

II Standard drug (1 mg/kg,

i.p.) + Pilocarpine

(1mg/kg, i.p.)

III Shilajit (25 mg/kg, p.o.) +

Pilocarpine (1mg/kg, i.p.)

IV Shilajit (50 mg/kg, p.o.) +

Pilocarpine (1mg/kg, i.p.)

Multiple dose study is conducted for duration of 15 days. Pilocarpine is administered on

15th day.

c. Apomorphine induced climbing behavior

GROUP

N=6

TREATMENT AND

DOSE

PARAMETERS

I Vehicle (1 ml/kg, p.o.) +

Apomorphine (3 mg/kg, 11

9.0s.c.)

Climbing behavior

II Standard drug (1 mg/kg,

i.p.) + Apomorphine (3

mg/kg, s.c.)

III Shilajit (25 mg/kg, p.o.) +

Apomorphine (3 mg/kg,

s.c.)

IV Shilajit (50 mg/kg, p.o.) +

Apomorphine (3 mg/kg,

s.c.)

Multiple dose study is conducted for duration of 15 days. Apomorphine is administered

on 15th day.

Statistical analysis:

The analysis of data will be done by using one - way ANOVA.

8.7 DOES THE STUDY REQUIRE ANY INVESTIGATION OR

INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER

HUMANS/ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.

Yes, the above study requires investigation to be done on the adult albino wistar

rats of either sex for the anti-epileptic and anti-psychotic study.

8.8 HAS ANIMAL ETHICAL COMMITTEE CLEARNACE BEEN OBTAINED

FROM YOUR INSTITUTION IN CASE?

The study has been referred to the ethical committee of the institution and

clearance has been obtained (Ref: SSCPT/IAEC. Clear 104/11-12, Dated: 30-11-

11) Enclosed copy. Annexure – I.

REFERENCES

1. Pandeya SN. A textbook of medicinal chemistry.3rd ed. Varanasi: SG Publisher;

2006. p. 158 (vol I).

2. Tripathi KD. Essentials of medical pharmacology. 6th ed. New Delhi: Jaypee

Brothers Medical Publishers; (P) Ltd. 2008. p. 401.

3. Munjal AM, Bharathi H, Ashok SK. Progress in neuroprotective strategies for

preventing epilepsy. Prog Neurobiol 2008;84:363-404.

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4. Moshi MJ, Kagshe GA, Mbuuambo ZH. Plants used to treat epilepsy by

Tanzanian traditional healers. J Ethnopharmacol 2005; 97:327 – 36.

5. Seizures and Epilepsy: Hope through Research. [Online]. 2011 Oct 31 [cited

2011 Nov 25]; Available from:

URL:http://www.ninds.nih.gov/disorders/epilepsy/epilepsy.html

6. Gelder, Michael. Psychiatry. New York: Oxford University Press Inc; 2005.

7. Mental health, poverty and development. [Online]. 2009 Jul 16 [cited 2011 Nov

25]; Available from:

URL:http://www.who.int/nmh/publications/discussion_paper_en.pdf

8. Mental disorders affect one in four people. [Online]. 2011 Oct 4 [cited 2011 Nov

25]; Available from:

URL:http://www.who.int/whr/2001/media_centre/press_release/en/index.html

9. Centrally connected. [Online]. 2006 Jul 15 [cited 2011 Nov 25]; Available from:

URL:http://www.expresspharmaonline.com/20060715/research01.shtml

10. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. Edinburgh:

Churchill Livingstone; 2003. p. 550.

11. Eugene W, Victor RG, Prasad DG, Petra K, Frauke M, Joyonto SF et al. Review

of Shilajit used in traditional Indian medicine. J Ethnopharmacol 2011;136:1-9.

12. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 41st ed. Vile-Parle (W)

Mumbai: Nirali Prakashan; 2008. p. 18.9.

13. Bhattacharya SK, Satyan KS, Chakrabarti A. Effect of trasina, an Ayurvedic

herbal formulation on pancreatic islet superoxide dismutase activity in

hyperglycemic rats. Indian J Exp Biol 1997;35: 297-9.

14. Prakash P, Pralhad P, Bhushan P. Ayurvedic treatment of obesity: A randomized

double - blind, placebo controlled clinical trial. J Ethnopharmacol 1990;29:1-11.

15. Jaiswal AK, Bhattacharya SK. Effects of Shilajit on memory, anxiety and brain

monoamines in rats. Indian J Pharmacol 1992;24: 12-17.

16. Kumar MR, Reddy AG, Anjaneyulu Y, Reddy GD. Oxidative stress induced

by lead and antioxidant potential of certain adaptogens in poultry. Toxicol Int

2010; 17:45-8.

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17. Ghosal S. Chemistry of Shilajit, an immunomodulatory ayurvedic rasayan. Pure

and applied chemistry. 1990; 62:1285-1288.

18. Shilajit A material medica monograph [Online]. 2004 Aug 30 [2011 Nov 25];

Available from:

URL:http:// www.jivaka.com/org/docs/paper/Talbert%20Paper.pdf

19. Bhattacharya SK, Bhattacharya A, Chakrabarti A. Adaptogenic activity of

siotone, a polyherbal formulation of ayurvedic rasayanas. Indian J Exp Biol

2000; 38:119-28.

20. Goel RK, Banerjee, Acharya SB. Anti-ulcerogenic and Anti-inflammatory

studies with Shilajit. J Ethnopharmacol 1990;29(1):95-103.

21. Achliya GS, Wadodkar SG, Dorle AK. Evaluation of CNS activity of Brahmi

ghrita. Indian J Pharmacol 2005;37:33-6.

22. Pinelli A, Trivulzio S, Zefinetti GC, Tofanetti O. Anti-convulsant effects by

reduced glutathione and related amino acids in rats treated with isoniazid. J

Toxicol 1988;48:103-07.

23. Maheshwar HG, Despande SV, Pramaod HJ. Anti-convulsant activity of fruits

of Terminalia chebulia retz. Against MES and PTZ induced seizures in rats. J

Herb Med Toxicol 2010;4:123-6.

24. Vogel HG. Drug Discovery and Evaluation. 2nd ed. New York: Springer Berlin

Heidelberg. 2002. p. 488, 531-7.

25. Finn M, Jassen A, Baskin P, Salamone JD. Tremulous characteristics of the

vacuous jaw movements induced by pilocarpine and ventrolateral striatal

dopamine depletions. Pharmacol Biochem Behav 1995;57: 243-9.

10 Signature of the candidate

11 Remarks of the Guide

The above information and literature has been extensively investigated and verified. The

present study will be carried out under my supervision and guidance.

14

12 12.1 Name and Designation of Guide

Mr. SYED MANSOOR AHMEDM. Pharm., (Ph.D)

Asst. Professor DEPARTMENT OF PHARMACOLOGY

12.2 Signature

12.3 Co-Guide ( If any) N.A.

12.4 Signature N.A.

13 13.1 Head of the Department Dr. THIPPESWAMY B.S.,M. Pharm., Ph. D.Professor & Head

DEPARTMENT OF PHARMACOLOGY

13.2 Signature

14 14.1 Remarks of the Principal The above mentioned information is correct and I recommend the same for approval.

14.2 Signature

Dr. S. Badami M.Pharm., Ph.D.Principal

Sree Siddaganga College of Pharmacy B. H. Road, Tumkur-572 102.

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